Chromatography (gas, liquid, electro-driven) is a powerful analytical tool that can separate, identify, and quantify multiple analytes in a single analysis. The principal components of a typical chromatographic system include (1) an inlet that provides the interface to transfer a sample mixture into the chromatographic (separation) column; (2) a separation column that separates the sample mixture into its individual components as these components are swept through the column by a mobile phase; (3) a mobile phase to provide a driving force to move solutes from one end of the column to the other, the separation being based on a combination of differential interactions between the components of the sample mixture, an immobilized liquid or solid material within the column (stationary phase) and mobile phase, and (4) a detector that detects and measures components as they exit the separation column at different times. The exit time of a component is defined as the “retention time (RT)” for that component. Some chromatographic methods are capable of separating more than two hundred components in a single analysis. However, for chromatographic methods involving large numbers of components, a significant amount of work is required to determine the RT of each individual component during chromatographic method development. Also significant is the amount of work needed to correlate data generated on multiple instruments performing the same analysis, even for a small number of components.
The problem of replication arises after the chromatographic method development is completed. There are several parameters that affect RT. These include column parameters (e.g., length, stationary phase, particle size, and inside diameter) as well as operating parameters for the chromatograph (e.g., mobile phase type and flow rate, column temperature, ramp rates, column outlet pressure, and stationary phase thickness). Whenever a chromatographic method is used subsequent to its development, it is virtually impossible to replicate all the parameters precisely enough to obtain exactly the same retention times as those observed initially. The cumulative effects of these small but finite differences in parameters usually lead to significant differences in RTs. As an example, when two “identical” gas chromatography (GC) systems were set up to run the same chromatographic method on the same pesticide samples, the RTs for specific solutes were different by 0.5 minutes for peaks eluting at 20 minutes.
Without exact replication, measured RTs do not match the RTs specified in the original chromatographic method or the computerized method files (including calibration and event tables) and can lead to misidentified peaks with grave consequences in applications such as forensic, clinical or environmental analysis. The need therefore exists for means to remove or easily compensate for these RT differences.
Prior Solutions and their Disadvantages
There exist two general ways improving the match of RTs over time and between one system and another: instrumental and calculational. Instrumental approaches seek to reduce differences in RTs by adjusting one or more instrumental parameters such as flow rate and temperature program rate. As a consequence of instrumental approaches, the actual retention times that are generated during analysis more closely match reference RTs.
In calculational approaches, the actual RT data are modified after the RT data are acquired. The most obvious and widely used calculational method for dealing with RT mismatch in a situation subsequent to that of the reference analysis is to re-run a mixture(s) containing all of the possible compounds to be analyzed to determine individual RTs in the new situation. This is a reasonable task for simple chromatographic methods with a small number of well-separated analytes. However, this process becomes much more difficult and time consuming as the number of analytes increases or when using different chromatograph configurations. In addition, this approach does not address the differences between the RTs obtained in a target chromatographic system and those in reference libraries and databases, nor does the approach help in visual or mathematical comparison of chromatographic data obtained on other instruments.
A popular “relative retention” calculational approach utilizes retention indices or Kovats indices that circumvent problems in getting the same retention time from instrument-to-instrument, column-to-column. This type of procedure converts the actual retention times of detected peaks into a number that is normalized to (usually) multiple reference compounds. The Kovats and other relative retention procedures are especially useful for comparing retention times to databases and libraries for identification of individual components. However, these procedures do not help in visual or mathematical comparison of chromatographic data obtained on other instruments, because the procedures adjust the retention times from the integration report and do not effect the plotted chromatographic data or the integrated (slice) data. In addition, most retention index calculations do not use a smoothed correction function, so the resulting indices rely on the accuracy and reproducibility of retention times of reference peaks that bracket the compounds of interest, and are therefore inherently less precise than when using a smoothed correction function.
Lantos et al. describe the application of a polynomial regression to facilitate comparison of retention data from two different GC pesticide databases (Lantos J. et al. “Validation of gas chromatographic databases for qualitative identification of active ingredients of pesticide residues” Principles and Practices of Method Validation 256:128-137, 2000). Although Lantos et al. had some success at correlating the data, this type of approach is fundamentally flawed. Specifically, the selected data used by the authors came from methods with significant method differences. Changes in stationary phase chemistry, temperatures and flows (outside the rules of method translation) that form the basis of the Lantos approach lead to changes in relative as well as absolute retention times of solutes. General mathematical approaches cannot correct for these changes. Note that in the Lantos reference, the corrected retention times of almost all (18 out of 23) of the peaks selected for listing deviated by more than 1%, with three exceeding 10%. The database searching time windows required for RT differences as high as in Lanto's reference (windows>1 min) would generate a prohibitively high number of hits, rendering the approach unusable. In addition, there is no accommodation for correcting peak response, scaling methods, or changing x-axis or y-axis units in Lanto's method.
An instrumental approach to matching GC retention times is described in U.S. Pat. No. 5,958,246 to Tipler et al. The Tipler technique somewhat improves the match in RTs between systems, but the technique is a very involved, time-consuming procedure and has proven to be limited in practical application.
A more recent and advantageous instrumental approach to solving these problems in GC is that of “retention time locking” (RTL). This technique, described in U.S. Pat. No. 5,987,959 to Klee et al., which is incorporated herein by reference, addresses the problem of matching RTs on multiple systems.
The Klee technique, referred to as the RTL I method hereafter, provides a method for automated matching of retention times obtained using a known chromatographic method having a defined set of column parameters and operating parameters to the retention times obtained using a new chromatographic method having a new set of column parameters, wherein the retention times of components separated in accordance with the new chromatographic method are matched to the retention times set forth in the known chromatographic method. A procedure is described to adjust head pressure to compensate for differences in a new versus the original column, carrier gas, and column outlet pressure of the known chromatographic method.
The use of the RTL I method to enhance identification of unknowns with the use of RT databases is described in U.S. Pat. No. 5,827,946 to Klee et al., which is incorporated herein by reference.
The RTL I method makes significant improvements in the degree to which retention times match between multiple systems and over time. All of the nine tasks listed above and more are improved with the use of the RTL I method. There are, however, shortcomings to the RTL I method. These shortcomings include:    1. While the RT of the locking peak is often very well matched (typically to within 0.005 min), the resolution of pressure adjustment (0.01 psi) is often insufficient to produce a match for the locking peak of better than 0.015 min for columns with inlet pressures below 5 psi. For columns with higher inlet pressures (greater than 20 psi), the match can usually be made to within 0.002 min.    2. Even if the RT of the locking peak is precisely matched, the peaks located at RTs significantly removed from the locking peak can still have significant RT differences from other columns, instruments, libraries, or databases. These differences can be large enough to cause misidentification of compounds and all of the other problems associated with RT differences.    3. In cases where method translation (described in U.S. Pat. No. 5,405,432 to Snyder et al.) is attempted using a column with a different phase ratio than the original one, the current forms of RTL cannot match RTs with as high quality. They deviate by amounts that are not easily predicted or compensated for experimentally.    4. Some analysts use methods with two columns of different types connected to a single injection port but to separate detectors. This approach allows for dual column identification. Only one of these columns can be locked using RTL, however, since there is only one pressure that can be set.